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A high‐throughput fluorescence polarization assay for the determination of Kd for soluble epoxide hydrolase inhibitors
Author(s) -
Ingraham Richard H.,
Kroe Rachel R.,
Morelock Maurice M.,
Proudfoot John R.,
Eldrup Anne B.,
Canada Keith,
Cardozo Mario,
Grygon Christine A.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.479.21
Subject(s) - epoxide hydrolase 2 , chemistry , fluorescence anisotropy , enzyme , active site , fluorescence , rhodamine , curcumin , biochemistry , high throughput screening , stereochemistry , combinatorial chemistry , membrane , physics , quantum mechanics
Soluble epoxide hydrolase (sEH) has been demonstrated to play a role in a number of physiological processes through its ability to convert epoxy fatty acids such as the highly active epoxyeicosatrienoic acids (EETs) into their less active vicinal diols. Because EETs exhibit potentially beneficial effects in various pathologies such as inflammation and hypertension, an sEH inhibitor could exert a therapeutic benefit by preventing their hydrolysis. We have developed a competitive binding fluorescence polarization screening assay for sEH inhibitors which utilizes a di‐alkylurea active site inhibitor linked to a rhodamine probe. The probe binds to various mammalian sEH enzymes with K d values in the low nanomolar range. Inhibitor compounds were initially fit to yield IC 50 values. However, using that simple fitting paradigm it was not possible to distinguish the affinities of more potent compounds. To address this, a sophisticated fitting algorithm was developed which enabled the use of more information from the binding curves and the calculation of K d values for the inhibitors.